Attachment of dissimilar materials is challenging due to stress concentrations that arise at their interface. The tendon-to-bone attachment site (enthesis) solves this mechanical problem using a functionally graded transitional tissue that includes spatial gradients in structure, extracellular matrix composition, and cell phenotype. This strong and tough attachment system is formed during fetal and postnatal timepoints under the regulation of molecular and biophysical cues. Unfortunately, this unique structure is not recreated after surgical repair and healing, leading to remarkably high failure rates. Therefore, our goal is to gain an understanding of tendon enthesis development in order to motivate regenerative strategies for enthesis repair. Our overall hypothesis is that temporal and spatial regulation of biochemical (TGF and Ihh) and biophysical (muscle force) cues are necessary to drive the development of a functional enthesis and for regeneration after injury. Our hypothesis is motivated by our recent work, which showed that embryonic tendon-to-bone attachment initiates from a distinct population of progenitor cells and postnatal enthesis maturation and mineralization is driven by Indian hedgehog signaling and muscle loading. It remains unclear, however, how these cell populations at the developing attachment regulate the formation of a mature enthesis. Therefore, Aim 1 will determine the lineage (or lineages) of the cells that compose the mature enthesis, starting at embryonic timepoints and progressing through skeletal maturity. Aim 2 will determine the molecular and mechanical regulation of the enthesis cell phenotype(s), focusing on TGF for early developmental events and Ihh signaling for later mineralization events. In Aim 3, we will determine the role of enthesis cell lineages and molecular signaling for repair and regeneration of the injured enthesis, focusing on cells responsible for Ihh signaling at the enthesis. These aims will allow us to identify and characterize the cells that populate the enthesis and to uncover the molecular and biophysical sequence of events that is required for their differentiation. Results will have a direct impact on future cell- and growth factor-based regenerative strategies for tendon-to-bone repair.